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GASTRIC ACID SECRETION: CATION BINDING IN H,K-ATPASE

胃酸分泌：H,K-ATP酶中的阳离子结合

基本信息

批准号：
6164515
负责人：
EDWIN C RABON
金额：
$ 18.89万
依托单位：
TULANE UNIVERSITY OF LOUISIANA
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-08-01 至 2001-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6164515
关键词：
active sites chemical binding chimeric proteins disulfide bond enzyme activity enzyme inhibitors enzyme mechanism enzyme structure gastric acid hydrogen potassium exchanging ATPase protein structure function secretion site directed mutagenesis

项目摘要

The gastric H,K-ATPase provides the enzymatic basis for acid secretion in the stomach. For the pathology leading to gastric ulcer diseases, the most efficacious treatment is the suppression of stomach acid with concomitant H pylori eradication. Though gastric and duodenal ulcers have historically been treated with H-2 blockers, future pylori eradication. Though gastric and duodenal ulcers have historically been treated with H-2 blockers, future alternatives such as that provided by the acid-activated thiol reagent, Omeprazole, may increasingly rely on the direct inhibition or modification of the H,K-ATPase. Reversible inhibitors, K plus competitive ligands of the H, K-ATPase, appear to interact at or near the cation binding domain. MDPQ, a novel fluorescent probe from this reversible class of pump inhibitors has been used to investigate the H,K-ATPase function at an inhibitor domain present in both the intact ATPase and a membrane preparation obtained by tryptic digestion of the enzyme. Insights gained of the cation binding domain in these preparations may aid in the design of future pharmacology, exploiting key features of pump function and structure. This work helps define an area of potential clinical relevance aimed at establishing the H, K-ATPase as a therapeutic target in ulcer disease. Progress will depend on the advance of knowledge of the H,K-ATPase structure and its relationship to function. A fundamental issue of this proposal is the elucidation of the structural basis of cation selectivity in Ion pumps. This focus is appropriate because the P type ATPase pumps have discriminate specificity and stoichiometry of cation transport whose structural basis is not evident from inspections of the 1 degree structure or comparisons of sequence homology between these pumps. The proposal provides a strategy to identify domains and residues within those domains that contribute to cation binding and ion translocation. It utilizes complementary strategies involving biochemical and molecular biological methodologies where residues within those domains that contribute to cation binding and ion translocation. It utilizes complementary strategies involving biochemical and molecular biological methodologies where residues implicated in studies of the native H, K-ATPase will provide a rational basis for refined structure-function studies utilizing site specific mutations. Together these studies will identify structural features of domains within the alpha and beta-subunits of the H,K-ATPase important for cation binding and will define residues essential for cation binding. This proposal focuses on the identification of charged residues within the transmembrane domain that are necessary for cation binding and the elucidation of the contribution of the beta-subunit to cation binding.

胃H，K-ATP酶为胃酸分泌提供酶基础，胃对于导致胃溃疡疾病的病理，有效的治疗是抑制胃酸，幽门螺杆菌根除。虽然胃和十二指肠溃疡在历史上接受过H-2阻断剂治疗，将来根除幽门虽然胃和十二指肠溃疡历来用H-2阻滞剂治疗，未来的替代物，例如由酸活化的硫醇提供的替代物，试剂，奥美拉唑，可能越来越依赖于直接抑制或 H，K-ATP酶的修饰。可逆抑制剂，K+竞争性 H，K-ATP酶的配体似乎在阳离子处或附近相互作用结合域 MDPQ，一种新型的荧光探针，的泵抑制剂已被用来研究H，K-ATP酶的功能，存在于完整的ATP酶和膜中的抑制剂结构域通过胰蛋白酶消化酶而获得的制剂。获得的见解这些制剂中阳离子结合结构域的含量可能有助于设计未来的药理学，开发泵功能的关键特征，结构这项工作有助于确定一个潜在的临床相关性领域目的是将H，K-ATP酶作为溃疡的治疗靶点疾病进步将取决于对人类的认识的进步。 H，K-ATP酶的结构及其与功能的关系这一建议的一个基本问题是阐明结构离子泵中阳离子选择性的基础。这种侧重是恰当的因为P型ATP酶泵具有区分特异性，结构基础不明显的阳离子迁移的化学计量从1度结构的检查或序列的比较这些泵之间的同源性。该提案提供了一项战略，结构域和那些结构域内有助于阳离子结合的残基和离子移位。它利用互补战略，生物化学和分子生物学方法，那些有助于阳离子结合和离子移位的结构域。它利用生物化学和分子生物学的互补策略，生物学方法，其中涉及的残留物的研究， H，K-ATPase的研究为进一步完善其结构功能提供了理论依据利用位点特异性突变的研究。这些研究将鉴定α和β亚基内结构域的结构特征的H，K-ATP酶重要的阳离子结合，并将确定残基对阳离子结合至关重要。该提案的重点是查明跨膜结构域中的带电残基，阳离子结合和β-亚基贡献的阐明到阳离子结合。